During the past year, we have continued to identify the hypophysiotropic neurons, i.e., those which have connections with portal capillaries of the median eminence; and are, therefore, directly involved in the regulation of the secretion of the anterior pituitary. To identify these neurons, we conducted retrograde labeling studies in combination with peptide immunocytochemistry. Fluoro-Gold was used as retrograde tracer, and the endogenous peptides were detected by fluorescence immunocytochemistry. Our observations indicate that the majority of thyrotropin-releasing hormone (TRH), enkephalin and corticotropin-releasing hormone (CRH) neurons are located in the paraventricular nucleus, while neurotensin, galanin and dopamine neurons are found in the arcuate nucleus. However, not each peptidergic neuron in these well-defined nuclei is hypophysiotropic. Approximately 30% of these neurons do not project to the median eminence but contact other neurons in the brain where hypophysiotropic factors function as neurotransmitters or neuromodulators. Interestingly, the endocrine milieu affects the connectivity between nerve terminals and capillaries in the median eminence. We have found that in lactating animals more than 90% of all dopaminergic neurons in the arcuate nucleus are connected to the portal system, while in cycling females only 65% of these neurons form contacts with the portal system. The retrograde label Fluoro-Gold provides an electron-dense substance in the cytoplasm providing a suitable technique to study the afferentation of hypophysiotropic neurons.